In chemoprevention of head and neck squamous carcinoma (HNSCC), new approaches must consider the sustained epigenetic effects of tobacco use on host molecular mechanisms and identification of novel agents with limited toxicity and acceptable therapeutic ratio. Smoking and nutrition have been identified as significant factors that contribute to carcinogenesis and prognosis of these cancers. Epigenetic CpG island methylation in the tumor promoter gene p16 is frequent in HNSCC and in the mucosa of smokers, premalignant dysplastic tissue and in non-cancerous tissue adjacent to proven malignancies suggesting an important role in early carcinogenesis and second primary malignancy. An important downstream target of p16 that may play a central role in HNSCC carcinogenesis is COX-2. COX-2 over expression is common in premalignant and malignant mucosa and is associated with smoking and chronic inflammation. When blocked, COX-2 decreases tumor growth and invasiveness. COX-2 is influenced by EGFR signaling and p16 inactivation which are commonly perturbed in HNSCC. We propose to identify frequency of gene methylation for p16 and 3 other candidate genes, gene expression alterations and related serum biomarkers associated with clinical outcome using our extensive retrospective tissue and clinical data resource. We will prospectively validate and expand these data and determine specific effects of a tailored smoking cessation program on modulation of identified biomarkers. A Phase II trial is proposed to determine if preoperative soy isoflavones can modulate these markers. Soy isoflavones have been shown to reverse p16 methylation, decrease COX-2 expression and modulate VEGF, IL6, tumor proliferation, angiogenesis and apoptosis. We hypothesize that suppressor gene hypermethvlation in tumor and adjacent non-tumor mucosa and associated molecular changes in downstream targets such as COX-2. VEGF. EGFR and IL6 are associated with clinical outcome and can be abrogated by soy isoflavone supplementation and smoking cessation. We will use pyrosequencing to measure methylation of p16 and three other candidate genes in pretreatment samples from both retrospective and prospective patients and correlate with outcome. We will determine expression of EGFR, COX-2, VEGF, p53 , p65,a6p4 integrin, HPV-16, Bcl-xL in tissue and levels of VEGF, IL6, HGF and 15-Ft2-isoprostane in serial serum and saliva samples from our prospective patients. This comprehensive proposal will investigate the molecular rationale for future soy isoflavone and smoking cessation prevention trials and identify intermediate molecular endpoints. These data will help define high risk patients according to biomarkers and smoking status who might benefit most from chemoprevention. Studies of upregulated gene profiles associated with tumor suppressor gene hypermethylation may identify new target genes for future study.